Cryptococcus neoformans is a major cause of morbidity and mortality in persons with T cell dysfunction, particularly AIDS. Mannoproteins (MP) are the primary component recognized by the anticryptococcal T cell-mediated immune response. In an effort to develop a vaccine, the PI has identified the molecular structure of MP and explored the mechanisms by which MP stimulate T-cell responses. Mannose receptors (MR) on dendritic cells (DC) bind to the mannosylated portion of MP resulting in antigen uptake, processing and presentation. However, there are problems with using MP as a vaccine. First, in murine models of cryptococcosis, vaccination with antigen preparations, including those containing MP, generally results in only partial protection. Second, MP neither trigger cytokine responses nor promote DC maturation. Thus, while mannosylation appears to result in very efficient delivery of antigen to DC, this alone is not sufficient to elicit a protective response. It is hypothesized that additional signals that promote DC maturation and the secretion of Th1-skewed cytokines are necessary for full protection to occur. This proposal focuses on boosting protective CD4+ T cell responses through the use of novel candidate vaccines that physically or chemically associate cryptococcal antigens with immune adjuvants. There are 3 specific aims: Aim 1: Create novel vaccine constructs by combining cryptococcal antigens with ligands that promote DC uptake, maturation and Th1 cytokine production. Native or recombinant cryptococcal antigens will be combined with ligands for the pathogen recognition receptors, dectin-1, TLR2, TLR7/8 and TLR9. Delivery systems include cryptococcal antigens directly conjugated to 2-glucans, Pam3CSK4, ssRNA, and CpG, as well as yeast glucan particles and microspheres loaded with antigens and ligands. Aim 2: Characterize the immune response following stimulation of DC populations with the cryptococcal antigen-containing constructs generated in aim 1. Four major responses will be studied: i) DC cytokine production, ii) DC maturation, iii) antigen uptake and localization within DC, and iv) antigen-specific presentation to T cells and the resulting T cell responses. Conventional and plasmacytoid DC from humans and mice will be studied, including DC from mice deficient in TLR2, TLR7, TLR9, and dectin-1. Aim 3: Test the immunogenicity and protective efficacy of candidate vaccines using in vivo mouse models of cryptococcosis. Promising candidate vaccines identified via the first two aims will be tested for their capacity to elicit immune responses in vivo and to protect against experimental cryptococcosis. Candidate vaccines will either be administered directly or as part of a DC vaccine. Completion of these studies over the next five years should add significantly to our knowledge of the molecular basis for the development of protective CD4+ T cell responses to C. neoformans and suggest novel approaches for vaccination against this often deadly mycosis. PUBLIC HEALTH RELEVANCE: Cryptococcosis has emerged as is a major cause of morbidity and mortality in immunosuppressed persons, particularly those with AIDS in whom it is the second most common cause of death worldwide. The studies proposed in this grant are focused upon developing a vaccine that will protect at risk individuals from acquiring cryptococcosis. As broad concepts are being studied, the knowledge gained should also be applicable to the development of vaccines against other infectious diseases.